Human herpesviruses remain a major public health concern and are responsible for many important human diseases. A critical means by which these viruses disseminate in vivo is cell-to-cell spread, a process that is highly relevant to vira pathogenesis. Spread of the infections by this mode allows efficient and rapid progeny virion dissemination, and also promotes immune evasion of neutralizing antibodies. In the case of herpes simplex virus (HSV), it is also critical for successful establishment of latent infections i neuronal cells. The long-term objective of this application is to elucidate the molecular mechanism of HSV cell-to-cell transmission so that better strategies and control measures can eventually be found to prevent the spread of infections. The focus of this application is glycoprotein E (gE), a membrane protein that has homologues among all alpha-herpesviruses and has long been known to be required for cell-to-cell spread, though the mechanism remains poorly understood. We have made substantial progress in understanding this molecule by investigating its protein-interaction network. Our studies have revealed a striking, coordinated assembly of three tegument proteins (UL11, UL16 and UL21) onto the cytoplasmic tail of gE, which leads to the formation of a functional complex required for virus-induced cell-cell fusion (syncytia formation), a special form of cell-to-cell spread. To pursue further clues of how gE works, we have designed three specific aims. In the first aim, we propose an unbiased genetic approach in which we will select for second-site repressors that restore the syncytia phenotype to mutants that have lost it because of the absence of the gE tail or its binding partners (e.g., deletion mutants gECT/gBsyn, UL11/gBsyn, UL16/gBsyn, and UL21/gBsyn). The Syn phenotype has a selective advantage because syncytial viruses can transmit viral genomes through the cell culture more quickly by causing cells to fuse. Because UL11, UL16 and UL21 are required for syncytia formation, this approach will be seeking a gain of function. One second-site suppressor of gECT/gBsyn has already been obtained. The second aim is to identify interactions between gE and other viral glycoproteins by means of a novel in vivo assay. Preliminary data have revealed an interaction with gD, and we hypothesize that direct interactions with the viral fusion machinery is required for gE to promote cell-to-cell spread and syncytia formation. The third aim is to determine the mechanism of how gE traffics to cell junctions and the relevance of gE cell surface expression for cell-to-cell spread. It has long been known that gE redistributes to cell junctions late in HSV infection, and this trafficking property s critical for gE to promote sorting of virions to lateral cell junctions, but details of the mechanim are not clear. These three aims will substantially advance our understanding of the molecular mechanisms of gE in HSV cell-to-cell transmission, which may eventually lead to discovery of novel targets for antiviral drugs that are needed for treatment of HSV infection.